Invasive aspergillosis (IA), caused predominantly by Aspergillus fumigatus, is the most common cause of fungal associated mortality in patients with chronic and severe immunosuppression caused by hematological cancers, transplantation, prolonged corticosteroid therapy, and genetic immunodeficiencies. IA mortality rates of 50% are common in heavily immunosuppressed leukemia patients and transplant recipients. No new classes of antifungals have been commercialized since 2001. The ineffectiveness of existing antifungal drugs warrants new therapeutic strategies. Cancer cells frequently express checkpoint proteins, such as PD-1 and CTLA-4, to evade attack by the immune system. Antibodies that target these proteins have shown much promise in treating certain cancers. Importantly, checkpoint blockade approaches to treating life-threatening IA infections that are unresponsive to conventional therapy have not yet been evaluated. We hypothesize that anti?PD-1 and/or anti? CTLA-4 checkpoint blockade, either alone or in combination with conventional antifungal agents, will be beneficial in an immunosuppressed murine model of invasive pulmonary aspergillosis (IPA). To test this hypothesis, we will perform the following Specific Aims: Specific Aim 1: Determine the efficacy of immune checkpoint blockade as a treatment for IPA. Our working hypothesis for this aim is that checkpoint blockade will be beneficial in IPA. 5 106 A. fumigatus conidia will be delivered to the lungs via intranasal administration in 8-week-old female BALB/c mice immunosuppressed with cyclophosphamide and cortisone acetate as per our previous studies. Starting 6 h after infection, mice will be given a daily intraperitoneal injection for 8 days, of either saline, anti?CTLA-4(100 ?g), anti?PD-1(250 ?g), or a combination of both checkpoint inhibitors (CPI?s). Intraperitoneal injection with Isotype IgG antibodies will serve as controls. The experiments will be terminated on day 9. We will assess the effects of CPI?s by measuring mouse survival, pulmonary fungal burden as assessed by both histology and quantitative PCR, and pulmonary and serum mRNA and protein levels, of Th1-signature cytokines (IFN-?, IL-6, and TNF-?, that are important in host responses in IPA) and Th2 cytokines (IL-4 and IL-10, that are associated with negative outcomes in IPA). Specific Aim 2: Determine the efficacy of checkpoint blockade along with antifungal therapy to treat IPA. Our working hypothesis for this aim is that CPI when administered in combination with antiifungals, will be more effective than CPI regimen alone. We will use the CPI regimen that elicits the best response in Aim 1 along with 1 of 2 antifungal drugs that have anti-Aspergillus activity, caspofungin (an echinocandin with immune enhancing properties) and posaconazole (a triazole with immunological neutral effects). We hypothesize that the combination of CPI and caspofungin would result in a more effective immune mediated clearance compared to CPI alone or compared to CPI plus posaconazole. This innovative preclinical study will determine if checkpoint blockade can serve as a novel strategy to treat life threatening opportunistic fungal infections.